In order to characterize patterns of global transcriptional deregulation in primary colon carcinomas, we performed gene expression profiling of some 300 rectal and colon carcinomas, and, as expected, identified comprehensive lists of deregulated genes. Genes that are upregulated are potentially required for the viability of colorectal cancer cells, and can therefore be considered oncogenes. Our systematic comparison of colon and rectal carcinomas also revealed a significant overlap of genomic imbalances and transcriptional deregulation, including activation of the Wnt/beta-catenin signaling cascade, suggesting similar pathogenic pathways. The functional validation of novel targets was performed in cell lines established from colorectal carcinomas that recapitulate the genomic and gene expression changes that we have previously observed in primary colorectal cancers (CRC). We used a combined functional genomics and systems biology approach to identify such anti-CRC targets. Genes that are highly overexpressed in tumor cells can be required for tumor cell survival, and have the potential to be selective therapeutic targets. In an attempt to identify such targets, we assesd the consequences of RNAi-mediated silencing of overexpressed genes that were selected from 300 gene expression profiles from colorectal cancers (CRC) and matched normal mucosa. In order to identify credible models for in-depth functional analysis, we first confirmed the overexpression of these genes in 25 different CRC cell lines. We then identified five candidate genes based on how profoundly they reduced the viability of CRC cell lines when silenced with either siRNAs or shRNAs; the genes are HMGA1, TACSTD2, RRM2, RPS2, and NOL5A. These genes were further studied by systematic analysis of comprehensive gene expression profiles generated following siRNA-mediated silencing. Exploration of these RNAi-specific gene expression signatures allowed the identification of the functional space in which the five genes operate, and showed enrichment for cancer specific signaling pathways, some known to be involved in CRC. By comparing the expression of the RNAi signature genes with their respective expression levels in an independent set of primary rectal carcinomas we could recapitulate these defined RNAi signatures, therefore establishing the biological relevance of our observations. This strategy identified the signaling pathways that are affected by the prominent oncogenes HMGA1 and TACSTD2, established a yet unknown link between RRM2 and PLK1, and identified RPS2 and NOL5A as promising potential therapeutic targets in CRC. Broad chromosomal copy number alterations -aneuploidy- define the genomic landscape of cancer cells. Chromosome 13 is one of the most commonly amplified chromosomes in CRC; in fact, its gain is specific for this tumor entity. However, it is difficult to identify the drivers of such recurrent whole arm amplifications. In collaborating with Dr. Natasah Caplen, we therefore conducted a systematic analysis by integrating genomic copy number changes and gene expression profiles. This analysis revealed highly overexpressed genes mapping to localized amplicons on chromosome 13, including NUPL1, LNX2, POLR1D, POMP, SLC7A1, DIS3, KLF5 and GPR180. RNAi-mediated silencing of these eight candidates resulted in a profound reduction of cellular viability. The functional space of the genes was then established by global expression profiling after RNAi exposure. One candidate, LNX2, not previously known as an oncogene, was involved in regulating NOTCH signaling. Silencing LNX2 reduced NOTCH levels but also downregulated the transcription factor TCF7L2 and markedly reduced Wnt signaling. LNX2 overexpression and chromosome 13 amplification therefore constitutively activates the Wnt pathway, offering evidence of an aberrant NOTCH-Wnt axis in CRC. In order to identify further candidates involved in the survival of CRC cells, we performed a functional screen using siRNAs that target genes involved in regulating apoptosis. This study revealed that inhibition of CASP8AP2/FLASH significantly reduced viability of CRC cell lines. Gene expression profiling after silencing revealed that this effect is mediated by altering the transcription of several members of replication-dependent histone genes as a result of the expression of the non-canonical polyA variants of these transcripts. In addition, the loss of function signature was enriched for targets of the NFkB and MYC transcription factors. Silencing also resulted in a significant increase of the tumor suppressor gene NEFH, hence generating biological relevance of its function. Repeating these experiments as a time course revealed a continuous gene expression signature of cell death.